The detection and quantification of microorganisms and viruses and biological molecules such as nucleic acids and proteins has important implications in many fields, including the diagnosis of human disease and infection and in food and environmental testing. Additionally, the detection or quantification of specific biomolecules from tissue, sputum, urine, blood, semen, saliva and other biological materials plays an increasingly important role in various fields including, without limitation, the identification of criminal suspects and in paternity testing.
Currently such testing procedures and assays commonly utilize nucleic acids or proteins as molecular probes with which to identify the presence of specific organisms, tissues, or molecules in a given sample. For example, nucleic acid probes and proteins such as antibodies can be "labeled", or linked to a detectable moiety such as a radionuclide, an enzyme (or enzyme substrate) capable of participating in a predetermined chemical reaction which can be independently monitored, or a fluorescent, luminescent or chemiluminescent compound. When the labeled probe molecule is exposed to a specific analyte under conditions allowing the probe and analyte to associate, the amount of associated label is correlated to the amount of analyte originally present in the sample.
While both nucleic acids and antibodies can be used as molecular probes, one of these types of probes may be more suitable than the other in a given application. For example, single-stranded nucleic acids are most often used in methods to detect target nucleic acids in a sample having nucleotide sequences complementary to that of the probe.
By a nucleotide sequence is meant an order of consecutive nucleotides (e.g., the phosphate esters of adenosine (A), thymidine (T), cytosine (C), guanadine (G), inosine (I) and uracil (U)) comprising a single-stranded nucleic acid, conventionally described from the 5' terminus to the 3' terminus of the subject nucleic acid strand. Under appropriate reaction conditions a single-stranded nucleic acid may hybridize with another single-stranded nucleic acid to form a double-stranded helical structure held together by hydrogen bonds between pairs of complementary bases on opposing strands. Generally in such a structure, A is hydrogen bonded to T or U, while G or I are hydrogen bonded to C (although occasional mismatches may occur without causing strand separation); in such a case each nucleic acid strand is said to be complementary to the other nucleic acid strand. Thus, nucleic acid probes are most commonly used to detect other nucleic acids in a target region of nucleotide sequence complementarity.
Because of the sequence-specific nature of nucleic acid hybridization, and because of evolutionary divergence of species at the nucleotide sequence level, nucleic acid probes are often more suitable for detecting a given species of organism than are antibody probes. Nucleic acid probes can be exquisitely sensitive. For example, small amounts of target nucleic acids can be amplified using methods such as the polymerase chain reaction (e.g., EPO 0 200 362) and transcription-based amplification systems (e.g., WO 91/01384, WO 93/22461 and WO 94/03472), and can then be detected using specific oligonucleotide probes. Nucleic acid probes can also effectively distinguish slight differences (sometimes a single mismatch) between versions of the same gene or nucleotide sequence, which can permit diagnostic screening for genetic defects or mutations. Nucleic acid probes can be used in the identification of a particular individual within a species, for example to discount or even identify a criminal suspect by screening specimens of his or her nucleic acids.
Antibodies also remain effective tools for the identification and detection of specific cells, viruses, and proteins. Immunodiagnostic methods can thus be used to test for the presence of a specific disease or infection state, genetic defect, or physiological condition connected with the specific antigen or substance sought to be detected.
The majority of assay methods employing nucleic acids and/or antibodies utilize a physical binding step in order to separate the probe:analyte complex from unbound probe. These assay methods are called "heterogeneous" assays. By "probe:analyte complex" or "probe:analyte conjugate" is meant a specifically-associated molecular species containing at least one antibody or nucleic acid probe molecule (preferably labeled with a detectable moiety or atom) in stable association with at least one analyte molecule. An analyte molecule is a molecular species such as a nucleic acid or antigen sought to be detected, quantitated and/or identified.
A "hybrid" or "probe:analyte hybrid" is a probe:analyte complex in which probe and analyte are both nucleic acids.
Assay methods utilizing a physical separation step may employ a solid phase matrix, such as glass, minerals or polymeric materials in the separation process. The separation may involve preferentially binding the probe:analyte complex to the solid phase matrix while allowing the unassociated probe molecules to remain in a liquid phase. In such a case the amount of probe bound to the solid phase support after a washing step is proportional to the amount of analyte in the sample. Alternatively, the assay may involve preferentially binding the unassociated probe while leaving the probe:analyte complex to remain in the liquid phase; in this case the amount of probe in the liquid phase, again after a washing step, is proportional to the amount of analyte in the original sample. When the probe is a nucleic acid or oligonucleotide the solid support can include, without limitation, an adsorbent such as hydroxylapatite, a polycationic moiety, a hydrophobic or "reverse phase" material, an ion-exchange matrix such as DEAE, a gel filtration matrix, or a combination of one or more of these solid phase materials. In the case of media such as gel filtration, the separation is not due to binding of the oligonucleotide but is caused by molecular sieving of differently sized or shaped molecules.
A heterogeneous assay method may also involve binding the probe to a solid phase matrix prior to addition of a sample suspected of containing the analyte of interest. The sample can be contacted with the label under conditions which would cause the analyte of interest to be labeled, if present in the sample mixture. The solid phase matrix may be derivatized or activated so that a covalent bond is formed between the probe and the matrix; alternatively, the probe may be bound to the matrix through strong non-covalent interactions, including, without limitation: ionic, hydrophobic, reverse-phase, immunobinding, chelating, and enzyme-substrate interactions. After the matrix-bound probe is exposed to the labeled analyte under conditions allowing the formation of a probe:analyte complex, the separation step is accomplished by washing the solid phase matrix free of any unbound labeled analyte. Conversely, the analyte can be bound to the solid phase matrix and contacted with labeled probe, with the excess free probe washed from the matrix before detection.
Yet another type of assay system is termed a "homogeneous assay"; such assays can generally take place in solution without a solid phase separation step and commonly exploit chemical differences between the free probe and the analyte:probe complex. An example of an assay system which can be used in a homogenous format is the hybridization protection assay (HPA) disclosed in U.S. Pat. No. 5,283,174, in which a probe is linked to a chemiluminescent moiety, contacted with a analyte and then subjected to selective chemical degradation or detectable change in stability under conditions which alter the chemiluminescent reagent bound to unhybridized probe without altering the chemiluminescent reagent bound to an analyte:probe conjugate. This patent enjoys common ownership with the present application and is incorporated by reference herein.
Competition assays in which a labeled probe or analyte competes for binding with its unlabeled analog, are also commonly used in a heterogeneous format. Depending on how such a system is designed, either the amount of bound, labeled probe or the amount of unbound, labeled probe can be correlated with the amount of analyte in a sample. However, such an assay can also be used in a homogeneous format without a physical separation step, or in a format incorporating elements of both a homogeneous and a heterogeneous assay.
The present invention may be used in a homogeneous format, a heterogeneous format, or a mixture of formats as outlined above. It relates to assays in which there is caused a reversible difference in the detectability of a label when coupled to a substance forming a complex or conjugate with the analyte, as opposed to when it is not so coupled. In a preferred aspect, the invention is concerned with means for establishing an environment in which a labeled substance is differentially detected in a complexed or bound form, as opposed to a "free" or unbound form.
A particular object of the present invention is to provide a method for detecting, identifying or measuring an analyte in which the detection method is based on the relationship of the label to its microenvironment, thus allowing the label to differentiate between a "bound" state and an "unbound" state, relative to the substance to which it is coupled. Another object of the present invention is to provide methods for altering the microenvironment of the labeled substance so as to exploit the ability of the label to so differentiate.
A further object of the invention is to provide an assay method which is simple and minimizes the number of operator steps necessary to obtain a result. Thus, the present invention is especially useful in a homogenous format wherein detection of the binding pair is accomplished without a physical separation step, although as mentioned above, the methods and compositions described herein may easily be adapted to include such a step if desired.
Still further, it is an object of the invention to provide a method of decreasing the amount of background signal in a diagnostic assay, thereby increasing the signal-to-noise ratio for the assay which may in turn allow the detection of smaller quantities of analyte in the original sample. Such an improvement in the sensitivity of diagnostic assays supplies a clear advantage in the prompt and accurate identification of, for example, trace amounts of pathogenetic bacteria, fungi, or virions in a patient specimen, which in turn can allow for more rapid and accurate diagnosis and treatment.
The following examples demonstrate the methods and compositions of the present invention utilizing light-emitting substances, specifically chemiluminescent and luminescent compounds, as labels. It will nevertheless be clear to those of ordinary skill in light of the present disclosure that the present invention is equally applicable to assay formats utilizing other types of detectable compounds as labeling agents, such as a fluorescent agent so long as the compound is susceptible to sequestration between a probe:analyte complex and a detergent micelle and one of these microenvironments quenches or inhibits the detectability of the label. In the present invention such sequestration occurs depending upon whether the probe to which the labeling compound is coupled is complexed with the analyte of interest or exists in an unbound or "free" state in the assay medium.
By "quench" is meant to prevent a labeling substance from being detected or detectable, and may act either directly or indirectly. In a preferred embodiment, quenching is accomplished through the use of a substance which prevents a triggering agent from reacting with a label (such as a chemiluminescent label), resulting in the inability of the quenched label to be made detectable as, for example, by the emmission of light. Alternatively, the quenching agent may interact with the label thereby absorbing energy emitted by the label, as is the case when, for example, the fluorescent label 1,5-IEDANS (Molecular Probes, Eugene, OR) interacts with the quencher moiety DABCYL (Molecular Probes, Eugene, OR) to prevent the emission of fluorescent light.
An assay format known as HPA (the hybridization protection assay), in which assay selectivity is based on the molecular microenvironment of the labeling reagent, is described (as mentioned above) in U.S. Pat. No. 5,283,174. In the HPA format an analyte-binding probe is joined to a labeling substance which undergoes differential degradation unless the labeled probe forms a stable complex with the analyte of interest. Addition of an oxidizing agent as hydrogen peroxide results in detectable chemiluminescence only from intact analyte associated label.
Another type of assay format in which the activity of the label is different depending on whether the substance to which it is coupled is bound to the analyte or not typically uses an enzyme as a label. The probe-coupled enzyme uses a substrate to elicit a colorometric, fluorometric or chemiluminescent signal, which is then detected. Such formats are described in U.S. Pat. Nos. 3,654,090, 3,817,837, and 4,190,496.
In U.S. Pat. No. 5,093,270 there is described a method which uses liposome vesicles to encapsulate marker molecules, including fluorescent and chemiluminescent molecules, which are subsequently released from the liposomes using water-miscible alcohols. U.S. Pat. Nos. 4,640,898 and 4,816,419 describe a competition assay for gentamicin utilizing charged SDS micelles to bind the oppositely charged gentamicin moiety of free fluorescein-labeled gentamycin; under the conditions described no such binding occurred when the gentamicin was conjugated with a specific antibody. The fluorescein label's intensity and changes in the polarity of emitted light were then detected.
Enhancement of fluorescent yield and changes of the polarization of the emitted light by surfactant micelles has been disclosed by Gratzel and Thomas, The Application of Fluorescent Techniques to the Study of Micellar Systems in 2 Modern Fluorescent Spectroscopy 169 (ed. E. L. Wehry 1976).